Biotin-avidin detection systems have been widely used in immunohistochemical staining procedures because of their versatility and the high binding affinity between biotin and avidin. Biotin can be conjugated to proteins such as antibodies, to lectins, and to nucleic acids. Avidin can be conjugated to fluorochromes and enzymes with minimal loss of activity.
However, a major drawback to using biotin-avidin detection systems in immunohistochemistry is the endogenous avidin-binding ability of many tissues and non-specific binding of biotin derivatized proteins and avidin conjugated proteins. Many tissues contain endogenous biotin-containing macromolecules which contribute to a specific but undesired binding. This specific binding can be blocked using a sequential addition of excess avidin followed by excess biotin (G. S. Wood and R. Warnke, J. Histochem. Cytochem. 29:1196, 1981). An additional problem is the non-specific binding of avidin, which is due to a number of poorly defined mechanisms which are probably related to ionic and or hydrophobic protein-protein or protein-substrate interactions. For example, avidin-horseradish peroxidase conjugates have been observed to bind to cellular components in liver and kidney, but not in skin, lymph node, or spleen. Heparin in mast cells treated with certain fixatives can bind avidin ionically. In addition, some commercial lots of avidin exhibit non-specific binding with chromatin while other lots do not.
To date, no one means of blocking the unwanted binding is suitable. For each type of unwanted binding, the cellular component or tissue type associated with the binding dictates the preferred method of eliminating the unwanted binding. See for example, Duhamel et al, Methods in Enzymology 184:201-207 (1990) which describes methods of blocking unwanted binding based on the source and tissue type of the binding.
Many of the methods used to block unwanted binding are non-systematic such as use of whole serum which limits the reproducibility of the method. A defined medium that could block any source of unwanted biotin-avidin binding irrespective of the tissue being immunostained would eliminate the major drawback to use of biotin-avidin detection systems in immunohistochemistry.